Cardiovascular disease, which includes coronary heart disease (CHD) and stroke, is the leading cause of death and disability in developed countries of the world. CVD is caused by the clogging of arteries. Major accepted risk factors for CVD include age, gender, hypertension, smoking, diabetes, elevated blood low density lipoprotein cholesterol (LDL-C), and decreased blood high density lipoprotein cholesterol (HDL-C).
HDL can protect against atherosclerosis in several ways. The most cited HDL function to protect against atherosclerosis is its participation in reverse cholesterol transport. During this process, HDL removes cholesterol from macrophages in the vessel wall, preventing the transformation of macrophages into foam cells, thereby preventing the build-up of fatty streaks and plaque in the vessel wall. The cholesterol that originated in the macrophages is then carried by HDL to the liver for ultimate excretion into the bile. HDL has additional attributes, including having anti-oxidant, anti-inflammatory, and anti-trhombic capabilities.
HDL can be measured by its cholesterol, total protein, or apoA-I levels. All of these HDL building constituents are distributed in various HDL particles of different size, lipid and protein composition, and surface charge. They also have different pathophysiological relevance. The many different functions of HDL are a result of the presence of specific lipids, proteins, and ratios of the two, as the specific lipids and proteins are unevenly distributed amongst the various HDL subclasses. Cells have several ways of controlling cholesterol level. A cell can use HDL for cholesterol uptake or cholesterol removal depending on the cell lipid level and on HDL lipid and protein composition. The different HDL particles interact with the different cellular-cholesterol removal pathways in a HDL subclass-specific way. The different HDL particles also participate differently in the anti-oxidation and anti-inflammation responses and other HDL functions depending on their size and lipid and protein composition.
Several methods have been developed for separating HDL subclasses in the last half century. Among these methods, two-dimensional gel electrophoresis has the highest resolution and creates the least number of artifacts. The two-dimensional gel-electrophoresis method, first published by C. Fielding in 1987, is commonly used to analyze proteins, as well as other molecules, such as nucleic acids. The analysis involves the separation of mixtures of biomolecules on the basis of two properties (e.g., charge and size) in two dimensions on two-dimensional gels. The two-dimensional gel electrophoresis method for HDL particle separation is based on the combination of two principles of electrophoretic separation: in the first dimension, the lipoprotein particles are separated by electrophoretic charge of HDL particles on agarose gel; in the second dimension, particles are further separated by size (based on molecular weight or lipoprotein-complex mass) on non-denaturing polyacrylamide gel Generally, the separated protein components are detected in the gel as discrete and uniquely positioned spots, recognized initially by monospecific first antibody against the protein of interest and followed by recognition of the first antibody with a second antibody monospecific to the first antibody and labeled with any of a variety of radio labels (such as fluorescent label chemiluminescent labels). Depending on the labeling, this method is specific and can be quantitative by virtue of the utilization of protein immuno-localization and image-analysis. As a result of employing the two-dimensional HDL separation method, different HDL particles have been associated with CVD risk in population-based cross-sectional studies and in drug intervention studies.
The two-dimensional gel electrophoresis technology is also useful in the diagnosis of the homozygous and heterozygous state for rare inherited HDL disorders, such as apoA-I/C-III/A-IV, apoA-I/C-III deficiency, isolated apoA-I deficiency, ABCA1 deficiency, LCAT deficiency, SRB1 deficiency, CETP deficiency, lipoprotein lipase deficiency, hepatic lipase deficiency, and endothelial lipase deficiency. Based on the scans generated using this technique, it has become possible to differentiate among the various HDL-subpopulation profiles and this also allows for very precise evaluation of the severity of CVD-risk in patients. Most of the patients who are carriers of one normal and one damaged gene (referred to as heterozygotes) of the above list also have reduced levels of HDL and premature CVD. Patients who are carriers of two damaged genes (referred to as homozygotes) of the above list generally have a very high risk for premature CHD. Patients affected with apoA-I deficiency have no HDL and have strikingly premature CHD. Whereas, patients affected with LCAT deficiency have only preβ-1 and α-4 HDL particles and are at moderate to high risk for CVD. Different mutations in the cholesterol ester transfer protein (CETP) can cause either increased or decreased CETP activity resulting in different changes in HDL particles. High CETP activity results in low levels of large α-1 and high levels of the small preβ-1 HDL particles. High CETP activity is associated with significant increased risk for CVD. Low CETP activity, which may be due to mutations in the gene encoding CETP or to effects of various drugs, causes high levels of α-1 HDL and low levels of preβ-1 HDL. This HDL subpopulation profile (high α-1 and low preβ-1) is associated with protection against CVD. Various mutations in the genes encoding lipoprotein-, hepatic-, and secretory-phospholipases can also be detected and recognized by their specific HDL subpopulation profile using this method.
Similar to HDL, LDL can also be separated into particles having different sizes; LDL is most commonly separated into small dense (sd) LDL and large LDL particles. It is proven and widely accepted in the lipoprotein field that sdLDL-C is more atherogenic than large LDL-C. The most common method for separating LDL by size is electrophoresis. The quantification of different LDL fractions is based on lipid staining in the gel, followed by density scanning, and then integrating the area under the curve. The major disadvantages of this method are that it is labor and time consuming, and it has poor resolution. A more recent method involves the use of a specific mixture of detergents for removing other lipoproteins, and then measuring cholesterol only in small dense LDL or sdLDL. This method is adaptable to high throughput automated analyzers, has been standardized, and is useful in the CVD risk assessment profile of the present invention.
Biomolecules, such as lipoproteins, separated via two-dimensional gel electrophoresis can be detected and identified with known immuno-detection techniques, such as immunoblotting: 1) for measuring specific protein component (i.e., Western blot analysis or Western blotting), 2) for measuring specific DNA component (i.e., Southern blot analysis or Southern blotting, and 3) for measuring specific RNA component (i.e., Northern blot analysis or Northern blotting). In Western blot analysis, proteins separated by electrophoresis in a polyacrylamide gel are transferred onto a membrane, followed by incubation with tagged first or tagged second antibodies. In Southern blot analysis, DNA fragments separated by electrophoresis on an agarose gel are transferred onto a membrane, incubated and hybridized with complementary (labeled) nucleic acid probes. In Northern blot analysis, RNA fragments separated by electrophoresis on an agarose gel are transferred onto one or more nitrocellulose membranes, incubated and detected with a suitable probe.
A typical Western blot analysis comprises the steps of preparing the protein samples, electrophoresis of the protein samples by one or two dimensional polyacrylamide gel, transferring the protein sample from the polyacrylamide gel to a membrane, blocking the membrane in a blocking solution (e.g., typically in PBS containing 3% BSA or 5% non-fat milk), followed by incubation of the membrane with tagged or plain monospecific first antibody diluted in PBS-Tween 20, and 3% BSA or 5% non-fat milk incubation mixture. After finishing incubation with the antibodies, unbound antibodies and tags are removed from the membrane by washing the membrane in the washing solution (PBS-Tween 20) several times until all residual nonspecific binding of tagged-antibody is removed. Often, plastic bags or open trays placed on a laboratory rocker or shaker are used to shake the incubation media during the incubation and washing cycles. Such processing means result in low efficiency and higher incidence of contamination of the immediate and larger environment. Further, these techniques present safety issues, due to the use of harmful toxins, including but not limited to use of mercury and/or radioactivity.